Can cryptic female choice prevent invasive hybridization in external fertilizing fish?

Polyandrous mating systems result in females mating with multiple males, generating opportunities for strong pre-mating and post-mating sexual selection. Polyandry also creates the potential for unintended matings and subsequent sperm competition with hybridizing species. Cryptic female choice allows females to bias paternity towards preferred males under sperm competition and may include conspecific sperm preference when under hybridization risk. The potential for hybridization becomes particularly important in context of invasive species that can novelly hybridize with natives, and by definition, have evolved allopatrically. We provide the first examination of conspecific sperm preference in a system of three species with the potential to hybridize: North American native Atlantic salmon (Salmo salar) and brook char (Salvelinus fontinalis), and invasive brown trout (Salmo trutta) from Europe. Using naturalized populations on the island of Newfoundland, we measured changes in sperm swimming performance, a known predictor of paternity, to determine the degree of modification in sperm swimming to female cues related to conspecific sperm preference. Compared to water alone, female ovarian fluid in general had a pronounced effect and changed sperm motility (by a mean of 53%) and swimming velocity (mean 30%), but not linearity (mean 6%). However, patterns in the degree of modification suggest there is no conspecific sperm preference in the North American populations. Furthermore, female cues from both native species tended to boost the sperm of invasive males more than their own. We conclude that cryptic female choice via ovarian fluid mediated sperm swimming modification is too weak in this system to prevent invasive hybridization and is likely insufficient to promote or maintain reproductive isolation between the native North American species.

Frequency-dependent viscosity of salmon ovarian fluid has biophysical implications for sperm-egg interactions.

Gamete-level sexual selection of externally fertilising species is usually achieved by modifying sperm behaviour with mechanisms that alter the chemical environment in which gametes perform. In fish, this can be accomplished through the ovarian fluid, a substance released with the eggs at spawning. While the biochemical effects of ovarian fluid in relation to sperm energetics have been investigated, the influence of the physical environment in which sperm compete remains poorly explored. Our objective was therefore to gain insights on the physical structure of this fluid and potential impacts on reproduction. Using soft-matter physics approaches of steady-state and oscillatory viscosity measurements, we subjected wild Atlantic salmon ovarian fluids to variable shear stresses and frequencies resembling those exerted by sperm swimming through the fluid near eggs. We show that this fluid, which in its relaxed state is a gel-like substance, displays a non-Newtonian viscoelastic and shear-thinning profile, where the viscosity decreases with increasing shear rates. We concurrently find that this fluid obeys the Cox-Merz rule below 7.6 Hz and infringes it above this level, thus indicating a shear-thickening phase where viscosity increases provided it is probed gently enough. This suggests the presence of a unique frequency-dependent structural network with relevant implications for sperm energetics and fertilisation dynamics. This article has an associated ECR Spotlight interview with Marco Graziano.

Evidence of hatch-time based growth compensation in the early life history of two salmonid fishes.

Initial body size can indicate quality within-species, with large size increasing the likelihood of survival. However, some populations or individuals may have body size disadvantages due to spatial/temporal differences in temperature, photoperiod, or food. Across-populations, animals often have locally adapted physiology to compensate for relatively poor environmental influences on development and growth, while within-population individual behavioral adjustments can increase food intake after periods of deprivation and provide opportunities to catch up (growth compensation). Previous work has shown that growth compensation should include within-population differences related to short growing seasons due to delayed hatch time. We tested the hypothesis that individual fish that hatch later grow faster than those that hatch earlier. The relative magnitude of such a response was compared with growth variation among populations. We sampled young of the year Arctic charr and brook trout from five rivers in northern Labrador. Daily increments from otoliths were used to back-calculate size to a common age and calculate growth rates. Supporting the hypothesis, older fish were not larger at capture than younger fish because animals that hatched later grew faster, which may indicate age-based growth compensation.

A synthesis of senescence predictions for indeterminate growth, and support from multiple tests in wild lake trout.

Senescence-the deterioration of functionality with age-varies widely across taxa in pattern and rate. Insights into why and how this variation occurs are hindered by the predominance of laboratory-focused research on short-lived model species with determinate growth. We synthesize evolutionary theories of senescence, highlight key information gaps and clarify predictions for species with low mortality and variable degrees of indeterminate growth. Lake trout are an ideal species to evaluate predictions in the wild. We monitored individual males from two populations (1976-2017) longitudinally for changes in adult mortality (actuarial senescence) and body condition (proxy for energy balance). A cross-sectional approach (2017) compared young (ages 4-10 years) and old (18-37 years) adults for (i) phenotypic performance in body condition, and semen quality-which is related to fertility under sperm competition (reproductive senescence)-and (ii) relative telomere length (potential proxy for cellular senescence). Adult growth in these particular populations is constrained by a simplified foodweb, and our data support predictions of negligible senescence when maximum size is only slightly larger than maturation size. Negative senescence (aka reverse senescence) may occur in other lake trout populations where diet shifts allow maximum sizes to greatly exceed maturation size.

Freezing ovarian fluid does not alter how it affects fish sperm swimming performance: creating a cryptic female choice 'spice rack' for use in split-ejaculate experimentation.

Cryptic female choice is often mediated chemically in external fertilizers by ovarian fluid (OF), which can change sperm swimming performance and bias paternity under sperm competition. Assessing cryptic female choice is hindered by the necessity of using fresh gametes and the short time window available to obtain diverse samples from wild animals. Using split-ejaculate experimental designs and samples from lake trout, brown trout and Atlantic salmon, we evaluated whether freezing OF alters the way in which it modifies sperm swimming. Sperm had improved swimming performance in the presence of OF over plain water, and the effect did not depend on whether the OF had previously been frozen. Freezing OF does not seem to alter the way it influences sperm. This allows the researcher to create a 'spice rack' of OF samples that can be used in studies on cryptic female choice, and opens up the possibility to compare animals mating under large spatial and temporal variability.

Assessing the potential for post-ejaculatory female choice in a polyandrous beach-spawning fish.

In species with limited opportunities for pre-ejaculatory sexual selection (behavioural components), post-ejaculatory mechanisms may provide opportunities for mate choice after gametes have been released. Recent evidence from a range of taxa has revealed that cryptic female choice (i.e., female-mediated differential fertilization bias), through chemical cues released with or from eggs, can differentially regulate the swimming characteristics of sperm from various males and ultimately determine male fertilization success under sperm competition. We assessed the potential role that such female-modulated chemical cues play in influencing sperm swimming characteristics in beach-spawning capelin (Mallotus villosus), an externally fertilizing fish that mates as couples (one male and one female) or threesomes (two males and one female) with presumably limited opportunities for pre-ejaculatory sexual selection. We assayed sperm swimming characteristics under varying doses and donor origins of egg cues and also examined the possibility of assortative mating based on body size. We found mating groups were not associated by size, larger males did not produce better quality ejaculates, and egg cues (regardless of dosage or donor identity) did not influence sperm swimming characteristics. Our findings suggest that intersexual pre-ejaculatory sexual selection and cryptic female choice mediated by female chemical cues are poorly developed in capelin, possibly due to unique natural selection constraints on reproduction.

Sexually selected traits are larger and more variable in male than female beach-spawning capelin, Mallotus villosus.

We evaluated whether morphological traits in capelin, Mallotus villosus, that appear to be sexually selected (pectoral fin, pelvic fin, anal fin, lateral ridge) were larger and more variable in males than females compared with naturally selected morphological traits (eyes, dorsal fin). Photographs were obtained of 136 capelin captured at two spawning sites and standardised measurements were taken of six morphological traits. Males had larger traits than females for a given body size and this was most pronounced in the traits thought to be sexually selected. Body size explained much of the variation in female traits but less variation in male traits, suggesting alternative selection pressures are involved. We suggest that larger male body size aids in endurance rivalry and sexually dimorphic traits help males to remain in physical contact with females while spawning on the beach.

Fish sperm competition in hatcheries and between wild and hatchery origin fish in nature.

Males compete pre- and post-mating to fertilize the maximum number of eggs. In polyandry, sperm competition occurs when sperm from two or more males compete to fertilize eggs from a female. Here we review how sperm competition from hatchery origin fish can cause loss of genetic variability in fish populations kept in captivity and in wild populations. In fish hatchery practices, sperm competition occurs in mass spawners that release gametes in tanks, and in artificial fertilizations when pooled semen is used. In mass spawnings sperm competition is difficult to tease apart from pre-mating competition and other post-mating selective mechanisms, whereas, studies focused on the use of pooled semen in different fish species have shown a clear relationship between sperm motility parameters and precedence in fertilization. In both situations, sperm competition will result in a loss of genetic variability that accumulates over generations, but hatchery protocols can be adjusted to mitigate it. Another source of concern regarding sperm competition for hatchery produced fish is the spatial and temporal overlap in spawning with wild individuals, either via aquaculture escapees or purposeful stocking programs. This may result in sperm competition between hatchery origin and wild males and impact natural populations. Our review suggests that in order to give every adult selected as broodstock an equal opportunity to produce offspring in captivity, mass spawning and the use of pooled semen should be limited.

The impact of a changing winter climate on the hatch phenology of one of North America's largest Atlantic salmon populations.

In northern temperate regions, some of the most dramatic effects of climate change are expected during the winter. Understanding how changing winter climates influence the seasonal timing of key life events is critical for implementing effective conservation strategies, especially for poikilotherms, whose physiology and development are particularly sensitive to changes in thermal environment. Four mathematical models are available to predict the timing of hatch and emergence in Atlantic salmon (Salmo salar); however, such models are only useful if the effect of temperature is both repeatable within and among maternal families, and predictable across variable temperature regimes. Using a split-brood experiment, we found the timing of hatch to be repeatable and predictable in Atlantic salmon from the Exploits River, one of the largest remaining wild populations in North America. Three of the available mathematical models under-estimated the timing of hatch by an average of 21-26 accumulated thermal units (ATU); however, we identified one model that provided reasonable estimates of hatch timing (average under-estimate 7 ATU) under the three incubation temperature regimes we tested. We applied this model to daily water temperature profiles from 2006-18 at four sites within the Exploits River watershed. Across all years and sites, the predicted dates at 50% hatch ranged between 8 March and 23 May, while predicted dates of 50% emergence ranged from 11 May to 13 June. By identifying the seasonal timing of these particularly vulnerable early life stages, this model can aid the implementation of conservation efforts for this ecologically and economically important population.

Impact of crude oil and the dispersant Corexit™ EC9500A on capelin (Mallotus villosus) embryo development.

Marine food webs are particularly vulnerable to oil spills if keystone species are impacted. To quantify lethal and sublethal toxicity in a key Holarctic forage fish, capelin embryos were exposed to Hibernia crude oil water accommodated fraction (WAF) produced at an oil-to-water ratio of 1:9 (v:v) and chemically-enhanced WAF (CEWAF) produced with the dispersant Corexit™ EC9500A at a dispersant-to-oil ratio of 1:10 (CEWAF H) or 1:50 (CEWAF L). Corexit alone yielded similar embryotoxicity to CEWAF. 10% CEWAF H, with total polycyclic aromatic hydrocarbons of 99.2 µg/L, decreased embryo survival following 10 h of exposure, while continual exposed to 1% CEWAF L decreased hatching and heart rates. Concentrations down to 0.1% CEWAF L increased in a dose-dependent manner the transcript level of cytochrome P4501a1 (cyp1a1) in hatched larvae. These data indicate that embryo-larval survival of capelin is likely at risk if an oil spill coincides in space and time with spawning.

Chemically-dispersed crude oil and dispersant affects sperm fertilizing ability, but not sperm swimming behaviour in capelin (Mallotus villosus).

The effects of petroleum aromatic hydrocarbons (PAHs) on the embryonic and larval life stages of teleosts have been extensively examined. However, very little work has been conducted on how spilled oil affects fish sperm and there is no related knowledge concerning oil dispersing agents. The objective of our study was to determine sperm performance of a teleost fish under direct exposure to different concentrations of WAF (water accommodated fraction) and CEWAF (chemically enhanced water accommodated fraction). Capelin sperm motility, swimming behaviour, and sperm fertilization ability were evaluated in a scenario of an oil spill untreated (WAF) and treated (CEWAF) with the dispersant Corexit® EC9500A. Sperm fertilizing ability was lower when exposed to CEWAF concentrations of 16.1 × 103 µg/L total petroleum hydrocarbons and 47.9 µg/L PAH, and when exposed to the dispersant alone. The mechanism responsible for this reduced fertilizing ability is not clear. However, it is not related to the percentage of motile sperm or sperm swimming behaviour, as these were unaffected. WAF did not alter sperm swimming characteristics nor the fertilizing ability. We suggest the dispersant rather than the dispersed oil is responsible for the decrease in the sperm fertilizing ability and hypothesize that the surfactants present in the dispersant affect sperm membrane functionality.

A novel sperm adaptation to evolutionary constraints on reproduction: Pre-ejaculatory sperm activation in the beach spawning capelin (Osmeridae).

Reproduction of external fertilizing vertebrates is typically constrained to either fresh or salt water, not both. For all studied amphibians and fishes, this constraint includes immotile sperm that are activated after ejaculation only by the specific chemistry of the fertilizing medium in which the species evolved (fresh, brackish, or salt water). No amphibians can reproduce in the sea. Although diadromous fishes may migrate between salt and fresh water, they are shackled to their natal environment for spawning in part because of sperm activation. Here, we report for the first time among all documented external fertilizing vertebrates, that in the absence of any external media, sperm are motile at ejaculation in a marine spawning fish (Osmeridae, capelin, Mallotus villosus). To illuminate why, we evaluated sperm behavior at different salinities in M. villosus as well as the related freshwater spawning anadromous rainbow smelt (Osmerus mordax). Surprisingly, sperm performance was superior in fresh water for both species. M. villosus spend their entire life at sea but our results show that their sperm are deactivated by sea water, suggesting a freshwater ancestry. By circumventing constraining water chemistry, we interpret the unique pre-ejaculatory sperm activation in this species as a novel adaptation that enables fertilization in the marine environment. These findings also contribute to understanding the persistence of anadromy, despite great energetic costs to adult fishes.

Stressful environments induce novel phenotypic variation: hierarchical reaction norms for sperm performance of a pervasive invader.

Genetic variation for phenotypic plasticity is ubiquitous and important. However, the scale of such variation including the relative variability present in reaction norms among different hierarchies of biological organization (e.g., individuals, populations, and closely related species) is unknown. Complicating interpretation is a trade-off in environmental scale. As plasticity can only be inferred over the range of environments tested, experiments focusing on fine tuned responses to normal or benign conditions may miss cryptic phenotypic variation expressed under novel or stressful environments. Here, we sought to discern the presence and shape of plasticity in the performance of brown trout sperm as a function of optimal to extremely stressful river pH, and demarcate if the reaction norm varies among genotypes. Our overarching goal was to determine if deteriorating environmental quality increases expressed variation among individuals. A more applied aim was to ascertain whether maintaining sperm performance over a wide pH range could help explain how brown trout are able to invade diverse river systems when transplanted outside of their native range. Individuals differed in their reaction norms of phenotypic expression of an important trait in response to environmental change. Cryptic variation was revealed under stressful conditions, evidenced through increasing among-individual variability. Importantly, data on population averages masked this variability in plasticity. In addition, canalized reaction norms in sperm swimming velocities of many individuals over a very large range in water chemistry may help explain why brown trout are able to colonize a wide variety of habitats.

Lazy males? Bioenergetic differences in energy acquisition and metabolism help to explain sexual size dimorphism in percids.

1. Differences in energy use between genders is a probable mechanism underlying sexual size dimorphism (SSD), but testing this hypothesis in the field has proven difficult. We evaluated this mechanism as an explanation for SSD in two North American percid species--walleye Sander vitreus and yellow perch Perca flavescens. 2. Data from 47 walleye and 67 yellow perch populations indicated that SSD is associated with the onset of maturation: typically, males of both species matured smaller and earlier and attained a smaller asymptotic size than females. Males also demonstrated equal (perch) or longer (walleye) reproductive life spans compared with females. 3. To examine whether reduced post-maturation growth in males was due to lower energy acquisition or higher reproductive costs we applied a contaminant mass-balance model combined with a bioenergetics model to estimate metabolic costs and food consumption of each sex. Mature males exhibited lower food consumption, metabolic costs and food conversion efficiencies compared with females. 4. We propose that slower growth in males at the onset of maturity is a result of decreased feeding activity to reduce predation risk. Our finding that SSD in percids is associated with the onset of maturity is supported by laboratory-based observations reported elsewhere, showing that changes in growth rate, consumption and food conversion efficiency were elicited by oestrogen (positive effects) or androgen (negative effects) exposure in P. flavescens and P. fluviatilis. 5. Researchers applying bioenergetic models for comparative studies across populations should use caution in applying bioenergetic models in the absence of information on population sex ratio and potential differences between the sexes in energetic parameters.